Our laboratory focuses on elucidating the coupling of forces, structure, and dynamics of biologically important macromolecules. The next challenge in structural biology is to understand the physics of interactions between molecules in aqueous solution. The ability to take advantage of the increasing number of protein and nucleic acid structures determined by x-ray crystallography and solution NMR will depend critically on this knowledge to understand the strength and specificity of interactions among biologically important macromolecules that control cellular function and to rationally design agents that can effectively compete with those specific interactions associated with disease. Our past results have shown that experimentally measured forces are very different from those predicted by current, conventionally accepted theories of intermolecular interactions. We have interpreted the observed forces as indicating the dominating contribution from water structuring energetics. Our research program uses osmotic stress and x-ray scattering to measure directly forces between biological macromolecules in macroscopic condensed arrays. We additionally measure changes in hydration accompanying specific recognition reactions of biologically important macromolecules in dilute solution. This is a first step toward linking our observation of dominating hydration forces between surfaces in condensed arrays to the interaction of individual molecules in solution. We previously showed there is a strong exclusion of salts and polar solutes from the nonpolar polymer, hydroxypropyl cellulose, due to hydration interactions. The biological consequences of the strength and ubiquity of these interactions means that the thermodynamics of assembly and recognition reactions that occur within the crowded environment of a cell will be very different from dilute solution. It also means that hydration or water structuring interactions will likely affect the distribution small solutes and salts around all macromolecules. In particular, the distribution of salts around charged macromolecules is likely quite different from predictions based on conventional theories. In order to test this we now have extensive measurements of forces between DNA helices with varying NaBr and TMABr (tetramethylammonium bromide) concentrations. Changes in the number of ions in the DNA phase as the distance between helices decreases can be calculated through the fundamental Gibbs-Duhem relationship of basic thermodynamics. Our initial analysis suggests that while the distribution of NaBr around DNA seems dominated by electrostatics, the distribution of TMABr salt appears to have an additional contribution from hydration repulsion. This reasoning may account for our previously puzzling results for the interactions between highly charged, double helical polysaccharide i-carrageenan. This polymer has charges bound to a hydrophobic backbone. The repulsion of salt from the backbone due to hydration forces seems at least as important as the electrostatic interactions at close distances. The ability to combine osmotic stress and fundamental thermodynamics to derive the distribution function of small solutes and salts around macromolecules in condensed arrays offers an unprecedented opportunity to formulate a physics for molecular interactions at close distances based on experimental observation. We have also uncovered an unusual feature of spermidine-induced assembly of DNA. The trivalent cation spermidine is an effective agent for condensing DNA into closely packed structures. At 2 mM spermidine, DNA helices assemble into hexagonally packed arrays with an interaxial spacing of 2.95 nm. With increasing concentration of spermidine, NaCl, or betaine glycine, we see a second phase with an interhelical spacing of 3.35 nm. This new peak is much broader than the reflection at 2.95 nm, indicative of a much shallower attractive energy minimum. The stronger energy minimum could reflect a highly ordered, intercorrelated arrays of helices with spermidine bound at localized sites to maximize attractive interactions. The weaker state could then reflect a movement of spermidine to other binding sites that are not as attractive or a loss of correlated binding between helices. We have continued investigating the role of water in the sequence specific recognition reaction of the restriction nuclease EcoRI. The exceptionally stringent specificity of this enzyme is a paradigm for recognition reactions. We measure the number of waters coupled to a binding reaction from the dependence of the binding free energy on water chemical potential or, equivalently, osmotic pressure. We have previously found that nonspecific complex of the protein sequesters some 110 more waters than the specific one and that this water is likely at the protein-DNA interface. We have further shown that the dissociation rate of the specific complex is also very sensitive to osmotic pressure requiring the net binding of 120-150 waters. During this past year we have investigated more closely the dependence of the number of water sequestered on DNA sequence. Sequences that differ by even a single base pair (star sequences) from the specific recognition sequence (GAATTC) EcoRI bind only slightly better than completely nonspecific sequences. This abrupt decrease in binding energy with even a single base pair change is accompanied by an abrupt increase in the water sequestered by the EcoRI-DNA complex. At low stresses the osmotic sensitivities of both the competitive equilibrium constants and dissociations rates of complexes with two star sequences and with nonspecific DNA are practically indistinguishable. At low osmotic stresses these complexes with noncognate sequences sequester about the same 110 waters as nonspecific complexes. At least some of this water, however, can be removed from these noncognate complexes by applying high enough osmotic pressures. Since equilibration times at high stresses are too long for convenient measurement of binding constants, we have used dissociation rates to determine loss of sequestered water. At higher pressures the behavior of star sequence complexes is strikingly different from the nonspecific complex. There is a clear correlation between the dissociation rate osmotic dependence and DNA sequence. The kinetic measurements show that the TAATTC star sequence complex loses about 90 of its initially sequestered waters at osmotic pressure of 150 atmospheres. The osmotic work required to removed these waters is about 4 Kcal/mole. Over the same range of pressures the complex with the weaker star site, CAATTC, only loses about 20-30 of its waters. There is no apparent loss of water from the nonspecific complex. The amount of water we are able to remove from a noncognate complex correlates with the with the ability of the enzyme to cleave the sequence. In principle, any sequestered water can be removed by applying high enough osmotic stress, but the work necessary to dehydrate complexes will naturally depend on resulting DNA-protein contacts. We have also started to investigate the correlation between water and binding strength of DNA complexes with lambda Cro repressor. The recognition stringency for this repressor is more typical of specific sequence DNA binding proteins. Single base pair changes from the optimal recognition sequence only reduce binding energies by a few kT. We have constructed a series of DNA fragments with consensus and altered Cro recognition sequences that span a range of 1000 in binding constant. Preliminary results for the osmotic pressure dependence of the dissociation rate indicate that as the binding energy decreases the number of sequestered waters increases. For approximately every factor 10 decrease in the binding constant an extra 8 water molecules are bound by the complex.